BackgroundCytokine storm (CS) presently lacks effective therapies. Activating cholinergic anti-inflammatory pathway (CAP) represents a promising strategy for suppressing excessive inflammation.PurposeQing-Fei-Pai-Du Decoction (QFPDD) has demonstrated clinical efficacy against COVID-19-associated inflammation. However, its mechanisms for counteracting CS are not fully elucidated.MethodsUsing an LPS-induced murine CS model, QFPDD's efficacy was assessed via survival analysis, cytokine measurement, and histopathology. Mechanistic interventions included vagotomy, splenectomy, α7nAChR knockout, norepinephrine (NE) depletion, and pathway inhibitors. The effects of QFPDD's spleen-distributed components on the expression of ChAT and α7nAChR were tested in splenic CD4+ T cells and macrophages, respectively. Transcriptomics, flow cytometry, ELISA, western blot, and PCR were used to profile the intrasplenic cholinergic signaling.ResultsQFPDD protects mice from LPS-induced CS. Bioinformatics analysis suggested that this protection may involve multiple QFPDD components acting on multiple points of CAP. The protective effect was abolished by splenectomy or α7nAChR knockout, but not vagotomy, indicating a spleen- and α7nAChR-dependent mechanism. Further investigations demonstrated that QFPDD systemically activated intrasplenic CAP signaling, including upregulating macrophages α7nAChR, increasing ChAT+CD4+ T cells (via inhibiting p38 MAPK activation), and elevating splenic contents of NE and acetylcholine (ACh). Notably, NE depletion reversed QFPDD's benefits. Spleen-distributed components (e.g., ephedrine, wogonin, naringin) directly upregulated the expression of ChAT in CD4+ T cells and/or α7nAChR in macrophages. Specifically, the most abundant component ephedrine promoted NE release, inhibited acetylcholinesterase (AChE), and increased ACh, thereby triggering the α7nAChR and suppressing inflammation.ConclusionQFPDD suppresses CS through the multi-component and multi-target synergistic activation of intrasplenic CAP signaling.

Noninfectious inflammatory diseases (NIDs), including neurological, cardiovascular, autoimmune and metabolic diseases, impose a heavy burden on the global medical system. Currently, the main drugs used in the treatment of NIDs are nonsteroidal anti-inflammatory drugs and glucocorticoids, which can alleviate the symptoms but are often accompanied by serious adverse effects. In contrast, active components of herbal medicine provide a new option for the treatment of NIDs because of their multi-targeting properties and low toxicity. With the complexity of inflammatory responses, the cholinergic anti-inflammatory pathway (CAP) can be a potential therapeutic pathway for NIDs. This review aims to elucidate the mechanisms by which the active components, extracts and formulas of Chinese herbal medicines modulate CAP for the treatment of NIDs. These therapeutic effects can be achieved by regulating key targets in the CAP, including the α7 nicotinic acetylcholine receptor, choline acetyltransferase, acetylcholinesterase and downstream signaling. These findings provide valuable hints for the development of new potential drugs targeting NIDs.Please cite this article as: Ming ST, Guo PY, Chen F, Zhang WD, Liu X. Chinese herbal medicines in the treatment of noninfectious inflammatory diseases: Cholinergic mechanisms and therapeutic potential. J Integr Med. 2026; Epub ahead of print.

Thermosetting materials exhibit advantages such as dimensional stability and elasticity but lack reprocessability due to their permanently cross-linked internal structure. Introducing a reversible cross-linked network endows materials with reprocessability but often compromises resilience and mechanical properties. Hence, it is still a significant challenge to develop recyclable elastomers with high elasticity as traditional thermosetting materials and remolding ability as traditional thermoplastic materials. Based on this, this work incorporates both reversible and irreversible cross-linked networks into a polyurethane system, constructing synergistic networks with distinct properties to achieve high elasticity and reprocessability simultaneously. In addition, by adjusting the proportion of the synergistic networks, the relationship between elasticity and reprocessability in different materials was investigated, revealing the synergistic effect between the dynamic network and the chemical cross-linked network. This work provides theoretical support for the design of elastomer materials that combine the high resilience of thermoset materials with the remolding ability of thermoplastic materials.

BACKGROUND & AIMS:Liver fibrosis, a common outcome of chronic liver diseases, is generally considered irreversible. Upregulation of clusterin (CLU), a secreted glycoprotein, occurs in several degenerative diseases and is suggested to have protective effects; however, its potential in treating or reversing liver fibrosis requires investigation. METHODS:Induction of CLU and its receptor low-density lipoprotein receptor-related protein 2 (LRP2) in fibrotic liver were determined using single-cell RNA sequencing and immunofluorescence. Its role and mechanism were examined in CLU knockout mice and mesothelial-specific LRP2 knockout mice. A peptide mimicking CLU was designed and verified. RESULTS:CLU expression was induced in pericentral senescent hepatocytes and then secreted during liver fibrosis in both patients and mice. CLU deletion exacerbated liver fibrosis, whereas treatment with recombinant CLU reduced fibrosis, suggesting that CLU induction feedback inhibits fibrosis progression. Mechanistically, LRP2, the receptor for CLU, is expressed specifically by liver mesothelial cells but not hepatic stellate cells. Recognition of CLU by LRP2 triggers mesothelial cell proliferation and migration from the liver surface into the pericentral region, followed by endocytosis of surrounding fibers, thus reducing liver fibrosis. Thereafter, we designed a 20-amino acid peptide mimicking CLU, named CLUtide, and determined its effect in resolving liver fibrosis, suggesting the considerable therapeutic potential. CONCLUSIONS:Pericentral senescent hepatocytes secrete CLU to induce the proliferation and migration of mesothelial cells to endocytose fibers by its receptor LRP2, thus reducing liver fibrosis. The engineered CLUtide mimicking CLU may be promising for resolving hepatic deposited fibers during liver fibrosis.

Manufacturing sufficient quantities of high-quality hepatocytes holds significant promise for the treatment of liver diseases and drug screening. Here, we developed a chemically defined, animal-free method for the large-scale production of human gallbladder epithelial cells (hGBECs) under good manufacturing practice conditions, enabling their clinical application. The cell products were characterized for growth ability, phenotype, freeze-thaw viability, genetic stability, biological contamination, tumorigenicity, and acute toxicity to ensure quality control and biological safety. We also provide a protocol for generating functional hepatocytes from hGBECs. The derived hepatocytes demonstrated typical liver functions, including albumin secretion, urea production, and drug metabolism. In addition, these cells were used in drug toxicity testing. We conducted further functional experiments on Cu2+ transport and alcohol metabolism. Transplantation of these cells in vivo was able to rescue mice from liver failure. This large-scale, convenient strategy for manufacturing hGBECs serves as a biobank for clinical applications and provides a valuable model for studying liver diseases.

The large-scale production of functional hepatocytes is critical for liver disease treatment, disease modeling, and drug development. Here, we present a protocol for isolating, expanding, and maintaining human gallbladder epithelial cells (hGBECs). We describe steps for characterizing hGBECs and their differentiation into hepatocytes. We then detail procedures to assess the functionality of the derived hepatocytes using key hepatic function assays.For complete details on the use and execution of this protocol, please refer to Chen et al.1

Cholangiocarcinoma (CCA) is a biologically diverse and highly aggressive cancer that arises from the biliary epithelium. It is typically divided into intrahepatic, perihilar, and distal types, each with distinct clinical behavior, genetic alterations, and therapeutic responses. Worldwide, the global incidence of CCA has risen steadily, accounting for nearly 15% of liver cancers and ∼3% of all gastrointestinal malignancies. CCA often presents at an advanced stage due to its silent onset and shows poor responsiveness to conventional chemotherapy, resulting in high mortality, accounting for ∼2% of cancer-related deaths worldwide. Risk factors include parasitic infections like liver flukes and chronic biliary diseases such as cholelithiasis and primary sclerosing cholangitis, although most cases have unknown origins. While early-stage patients may benefit from surgical resection or liver transplantation, these options are often not viable in advanced disease due to high relapse rates. In cases of unresectable or metastatic CCA, treatment remains difficult due to resistance and a lack of effective targeted therapies. This review systematically integrates the genomic, epigenetic, and signaling network mechanisms underlying CCA with their translational implications, providing a critical synthesis of the rapidly evolving field of targeted therapies, including recently approved Food and Drug Administration treatments and emerging novel agents. We specifically emphasize the key mechanisms of therapeutic resistance and corresponding strategies to overcome them, present an updated evaluation of vulnerabilities across distinct molecular subgroups, and explore the major challenges and future trajectories for advancing biomarker-driven precision medicine in CCA, thereby offering a forward-looking and clinically relevant perspective.

Ageing erodes human immunity, in part by reshaping the T cell repertoire, leading to increased vulnerability to infection, malignancy and vaccine failure 1–3 . Attempts to rejuvenate immune function have yielded only modest results and are limited by toxicity or lack of clinical feasibility 1,3–5 . Here we show that the liver can be transiently repurposed to restore age-diminished immune cues and improve T cell function in aged mice. These immune cues were found by performing multi-omic mapping across central and peripheral niches in young and aged animals, leading to the identification of Notch and Fms-like tyrosine kinase 3 ligand (FLT3L) pathways, together with interleukin-7 (IL-7) signalling, as declining with age. Delivery of mRNAs encoding Delta-like ligand 1 (DLL1), FLT3L and IL-7 to hepatocytes expanded common lymphoid progenitors, boosted de novo thymopoiesis without affecting haematopoietic stem cell (HSC) composition, and replenished T cells while enhancing dendritic cell abundance and function. Treatment with these mRNAs improved peptide vaccine responses and restored antitumour immunity in aged mice by increasing tumour-specific CD8 + infiltration and clonal diversity and synergizing with immune checkpoint blockade. These effects were reversible after dosing ceased and did not breach self-tolerance, in contrast to the inflammatory and autoimmune liabilities of recombinant cytokine treatments 6,7 . These findings underscore the promise of mRNA-based strategies for systemic immune modulation and highlight the potential of interventions aimed at preserving immune resilience in ageing populations.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

Development of functional recovery therapies is critical to reduce the global impact of stroke as the leading cause of long-term disability. Our previous studies found that acute-phase protein orosomucoid (ORM) could provide an up to 6h therapeutic time window to reduce infarct volume in acute ischemic stroke by improving endothelial function. However, its role in neurons and functional recovery post-stroke remains largely unknown. Here, we showed that exogenous ORM administration with initial injection at 0.5h (early) or 12h (delayed) post-MCAO daily for consecutive 7 days significantly decreased infarct area, improved motor and cognitive functional recovery, and promoted mitochondrial biogenesis after MCAO. While neuron-specific knockout of ORM2, a dominant subtype of ORM in the brain, produced opposite effects which could be rescued by exogenous ORM. In vitro, exogenous ORM protected SH-SY5Y cells from OGD-induced damage and promoted mitochondrial biogenesis, while endogenous ORM2 deficiency worsened these processes. Mechanistically, inactivation of CCR5 or AMPK eliminated the protective effects of ORM on neuronal damage and mitochondrial biogenesis. Taken together, our findings demonstrate that ORM, mainly ORM2, is an endogenous regulator of neuronal mitochondrial biogenesis by activating CCR5/AMPK signaling pathway, and might act as a potential therapeutic target for the functional recovery post-stroke.

There are presently no acknowledged therapeutic targets or official drugs for the treatment of muscle fatigue. The alpha7 nicotinic acetylcholine receptor (α7nAChR) is expressed in skeletal muscle, with an unknown role in muscle endurance. Here, we try to explore whether α7nAChR could act as a potential therapeutic target for the treatment of muscle fatigue. Results showed that nicotine and PNU-282987 (PNU), as nonspecific and specific agonists of α7nAChR, respectively, could both significantly increase C57BL6/J mice treadmill-running time in a time- and dose-dependent manner. The improvement effect of PNU on running time and ex vivo muscle fatigue index disappeared when α7nAChR deletion. RNA sequencing revealed that the differential mRNAs affected by PNU were enriched in glycolysis/gluconeogenesis signaling pathways. Further studies found that PNU treatment significantly elevates glycogen content and ATP level in the muscle tissues of α7nAChR +/+ mice but not α7nAChR -/- mice. α7nAChR activation specifically increased endogenous glycogen-targeting protein orosomucoid (ORM) expression both in vivo skeletal muscle tissues and in vitro C2C12 skeletal muscle cells. In ORM1 deficient mice, the positive effects of PNU on running time, glycogen and ATP content, as well as muscle fatigue index, were abolished. Therefore, the activation of α7nAChR could enhance muscle endurance via elevating endogenous anti-fatigue protein ORM and might act as a promising therapeutic strategy for the treatment of muscle fatigue.

Interleukin-6 (IL-6) is a pleiotropic cytokine and exerts its complex biological functions mainly through three different signal modes, called cis-, trans-, and cluster signaling. When IL-6 binds to its membrane or soluble receptors, the co-receptor gp130 is activated to initiate downstream signaling and induce the expression of target genes. In the liver, IL-6 can perform its anti-inflammatory activities to promote hepatocyte reprogramming and liver regeneration. On the contrary, IL-6 also exerts the pro-inflammatory functions to induce liver aging, fibrosis, steatosis, and carcinogenesis. However, understanding the roles and underlying mechanisms of IL-6 in liver physiological and pathological processes is still an ongoing process. So far, therapeutic agents against IL‑6, IL‑6 receptor (IL‑6R), IL-6-sIL-6R complex, or IL-6 downstream signal transducers have been developed, and determined to be effective in the intervention of inflammatory diseases and cancers. In this review, we summarized and highlighted the understanding of the double-edged effects of IL-6 in liver homeostasis, aging, inflammation, and chronic diseases, for better shifting the "negative" functions of IL-6 to the "beneficial" actions, and further discussed the potential therapeutic effects of targeting IL-6 signaling in the clinics.

At present, there are no official approved drugs for improving muscle endurance. Our previous research found acute phase protein orosomucoid (ORM) is an endogenous anti-fatigue protein, and macrolides antibiotics erythromycin can elevate ORM level to increase muscle bioenergetics and endurance parameters. Here, we further designed, synthesized and screened a new erythromycin derivative named HMS-01, which lost its antibacterial activity in vitro and in vivo. Data showed that HMS-01 could time- and dose-dependently prolong mice forced-swimming time and running time, and improve fatigue index in isolated soleus muscle. Moreover, HMS-01 treatment could increase the glycogen content, mitochondria number and function in liver and skeletal muscle, as well as ORM level in these tissues and sera. In Orm-deficient mice, the anti-fatigue and glycogen-elevation activity of HMS-01 disappeared. Therefore, HMS-01 might act as a promising small molecule drug targeting ORM to enhance muscle endurance.

目的 通过小分子化合物改变细胞命运,诱导人胆囊上皮细胞(hGBEC)分化为具有功能的肝细胞样细胞.方法 在基质胶中对原代hGBEC进行三维培养,生长培养基中添加B27添加剂、N2添加剂、N-乙酰半胱氨酸、表皮生长因子、肝细胞生长因子等.添加小分子化合物/蛋白因子对细胞进行诱导分化,筛选出关键作用因子.采用PCR、qPCR和免疫荧光染色检测干细胞标志物及肝细胞相关标志物的表达情况,通过脂肪BODIPY-493染色、糖原过碘酸希夫染色和白蛋白ELISA检测评估细胞的肝样功能.结果 三维培养的hGBEC表达造血干细胞抗原CD133、上皮细胞黏附分子、肝细胞核因子4α等肝脏干细胞和肝前体细胞标志物.TGF-β信号通路抑制剂和Notch信号通路抑制剂是诱导hGBEC分化的关键作用因子.分化条件下所得细胞表达肝细胞功能标志物α1-抗胰蛋白酶、细胞色素P4503A4、白蛋白和延胡索酰乙酰乙酸水解酶,可以贮存糖原,具有合成脂肪的能力,能够分泌白蛋白.结论 hGBEC可在体外长期培养,通过抑制TGF-β和Notch信号通路可初步诱导其分化为具有部分肝功能的肝细胞样细胞.

细胞工程旨在通过相关理论让学生掌握如何科学改造细胞以获取目的产物,注重培养学生的科研思维、解决问题的能力和实践操作.在以往授课过程中,教师发现多数学生对课程相关领域的知识储备有限.文章介绍了案例教学在细胞工程中的具体实施方案与授课效果,通过围绕"诱导型多能干细胞的获取与应用"这一主题,安排课前资料查阅、课堂回答讨论、教师答疑与总结等模块完成授课,最后通过问卷调查、成绩评测分析教学效果.新的教学模式激起了学生的学习热情,锻炼了学生的探索能力,培养了专业思维素养,并延续了学生对所学专业的兴趣.今后,教师可通过凝练专业知识结合拓展具体应用,继续在该课程中开展案例教学.

Objective To construct a culture system of human gallbladder organoids, successfully culture them in vitro and identify their characteristics. Methods Human gallbladder epithelial cells were isolated and embedded in matrix glue using a 3-dimensional culture system for 3-dimensional culture. The area, perimeter and morphology were observed during the growth of gallbladder organoids and the form factor was calculated. The expression of the stemness markers（CD133 and leucine rich repeat containing G protein coupled receptor 5 [LGR5]） and the bile duct epithelial cell markers（hepatocyte nuclear factor 1β[HNF1β], epithelial cell adhesion molecule [Ep CAM], cytokeratin 7, cytokeratin 19, sex determining region Y box 9 [SOX9]） of gallbladder organoids was detected by immunofluorescence staining. After adding small molecules CHIR-99021 and blebbistatin into the growth medium, 5-bromodeoxyuridinc（BrdU） incorporation assay was used to detect the proliferation characteristics of organoids. Results During the in vitro culture process of gallbladder organoids, the cell area was gradually increased, and there was a significant difference between the area on day 7 and day 1（P＜0.01）. The form factor was increased from 0.80（0.75, 0.84） on day 1 to 0.83（0.81, 0.85） on day 7（P＜0.01）, indicating that the organoids grew stably in vitro and tended to form a perfect circle. Immunofluorescence staining showed that the bile duct epithelial cell markers were expressed in organoids. After adding small molecules CHIR-99021 and blebbistatin into the medium, BrdU detection showed that the proliferation of the organoids was increased（P＜0.01）. Conclusion Human gallbladder organoids with proliferative ability can be successfully cultured in vitro, and it has the characteristics of bile duct epithelial cells and can be used for the research and modeling of biliary diseases.

以细胞内微丝束的观察来体现细胞骨架知识体系是生物医学本科层次开设的实验必修项目之一.文章以细胞骨架重要功能特点-支撑和维持细胞形态为出发,对细胞进行细胞松弛素B处理和恢复并结合两种染色方法来分析细胞的形态与细胞骨架间的关系.该实验的优化,有助于学生理解细胞骨架功能,取得了较好的教学效果.

Purpose: Cancer stem cells (CSCs) have been considered involving in tumorigenesis, local recurrence, and therapeutic drug resistance of hepatocellular carcinoma (HCC). To investi-gate novel and effective methods for targeting hepatic CSCs is crucial for a permanent cure of liver cancer. Methods: The expression level of SIRT1 was detected in CSCs of HCC tissues and cancer cell lines. Expression of CSC markers, the self-renewal and tumorigenic ability of liver CSCs were analyzed with SIRT1 inhibition. Cellular senescence-related markers were used to detect CSCs senescence after inhibition of SIRT1. Results: SIRT1 was highly expressed in CSCs of HCC cell lines and human HCC tissues. In vitro study revealed that decreasing of SIRT1 level significantly downregulated the stemness-associated genes of liver CSCs and reduced the CSC stemness properties. Also, down-regulated SIRT1 suppressed liver CSCs proliferation by decreasing their self-renewal abil-ities. Furthermore, CSCs with decreased SIRT1 expression showed limited tumorigenicity and formed smaller HCC tumor in vivo. And SIRT1 decreased CSCs became more suscep-tible to chemotherapeutic drugs. Mechanistically, SIRT1 decreased CSCs became senescence through the activation of p53-p21 and p16 pathway. The data further indicated that the tumor formed from SIRT1-knockdown CSCs exhibited higher senescence-associated beta-galactosidase (SA-beta-Gal) activity but lower proliferative capacity. Conclusion: Taken together, these findings pointed that induction of senescence in liver CSCs is an effective tumor suppression method for HCC, and SIRT1 may be served as a promising target for HCC treatment.